This application addresses the broad Challenge Area (15) Translational Science, 15-CA-103: Thyroid Cancer Cell Line Project which is in direct response to our recent discovery showing that 17 out of 40 "thyroid" cancer cell lines are either redundant or misidentified (i.e. of a different tumor lineage including melanoma and colon cancer). The remaining 23 lines are unique and are likely of thyroid origin based on the expression of a limited number of thyroid-specific genes. These cell lines, however, have not been genetically linked to tumors of origin or carefully characterized at a genetic or molecular level. This problem for the research community was reviewed at a recent NCI conference in which the Principal Investigators participated. The need for well- validated thyroid cancer cell lines to study mechanisms of cancer development and progression as well as discovery of novel therapeutic targets was emphasized. In this proposal, we will use three complementary strategies to provide a large panel of comprehensively characterized thyroid cancer cell lines representing primary and metastatic tumors of different histologic types and mutational subtypes (BRAF, RET/PTC, RAS, PIK3CA, AKT1, CTNNB1). We will focus on developing cell lines from poorly differentiated thyroid cancer as well as metastatic lesions that are more likely to harbor mutations that are not represented in the current panel of thyroid cancer cell lines. To establish new cell lines, we will use a parallel in vivo and in vitro approach to maximize our success rate. The new cell lines will be carefully characterized and compared to the corresponding tumor tissue of origin by short tandem repeat (STR) profiling to confirm their derivation. STR profiling and expression of thyroid-specific genes will be used investigate the origins of an additional 36 thyroid cell lines reported in the literature in order to assemble a complete panel of thyroid cancer cell lines. We will use expression arrays of authentic thyroid cancer cell lines to identify a signature that can be used to validate cell lines that have lost expression of thyroid-specific markers or from which patient DNA is unavailable. Finally, we will apply global molecular and genomic approaches with novel computational analyses to the new and existing thyroid cancer cell lines to uncover pathways important in thyroid cancer development and progression. All cell lines, as well as the genomic and gene expression data, will be made available to the research community through a central repository with the assistance of the NCI, and data will be accessible through the NCBI GEO database and UCSC genome browser. Feasibility and Impact: The generation of new cell lines from solid tumors is a challenging task. We propose to generate at least 10 new cell lines in a 12-15 month time period using parallel in vivo and in vitro approaches. There is significant expertise at the University of Colorado Cancer Center (UCCC) with a proven record of success in generating cell lines from solid cancers. We believe that this ambitious proposal is feasible within the two-year time frame because the unique collaboration between the UC Denver and MSKCC has all of these advanced technologies currently in place (new procedures to develop cell lines, gene expression platforms, mass spectrometry genotyping, CGH array analysis, mutational screen of key oncogenes and tumor suppressor genes and high throughput pathway activation analysis by Western blotting). A majority of the sample preparation and studies for the arrays and mutation screening will be performed by experienced personnel in well-equipped cores with rapid turn-around times. Furthermore, we are collaborating with nationally recognized bioinformatics and computational experts to apply novel computational biology strategies to identify important signaling pathways that will better define disease pathogenesis. Successful completion of these aims will provide approximately 40 well-characterized thyroid cancer cell lines for use by the research community to uncover mechanisms of thyroid cancer development, progression, metastatic potential and to serve as a platform for identification of novel therapeutic targets for advanced disease. Lay Summary: Thyroid cancer is the most common endocrine malignancy with an annual incidence of ~34,000. At least 300,000 people are living with a diagnosis of thyroid cancer in the United States, and more than 1,500 of them die each year from this disease. Permanent thyroid cancer cell lines derived from patient's cancers are critical to help us understand the causes of thyroid cancer, how the tumor grows and spreads, and how we can find new ways to treat advanced disease for which there is currently no cure. Recent studies by our research groups have shown that nearly half of the "thyroid" cancer cell lines in current use are not unique and many did not even originate from a thyroid cancer. In order to provide well characterized thyroid cancer cell lines for study by the research community, we will generate new thyroid cancer cell lines that can be linked back to the original tumor tissue. We will also develop approaches by which we can determine whether some of the existing thyroid cancer cell lines are authentic or not. We will develop genetic profiles of all the cancer cell lines, from which we will be able to characterize the abnormal signaling networks that may explain how these cancers develop. Once we know what makes these cancers grow, we will have a better understanding of how to treat them. With the help of the National Cancer Institute, we will make these cell lines widely available to the research community. PUBLIC HEALTH RELEVANCE: The generation of a well-characterized panel of thyroid cancer cell lines has significant relevance for public health as validated tools for the broader research community. Successful completion of the proposed aims will provide approximately 40 well-characterized thyroid cancer cell lines in a central repository for use by the research community to uncover mechanisms of thyroid cancer development, progression, metastatic potential and to serve as a platform for identification of novel therapeutic targets for many types of advanced cancer.